1. Field of the Invention
This invention relates to catalytically active enzymes and more particularly to immobilized enzymes deposited on inorganic materials. Enzymes usually have high molecular weights, can catalyze numerous specific chemical reactions, and techniques have been developed to increase their efficiency in use. These techniques were developed because the enzymes are generally water soluble and for this reason when used in solution, they can only be used batch-wise, since it is difficult to remove them from the reaction medium. The techniques developed were directed to the immobilization or fixing of the enzymes on substantially water insoluble supports without loss of activity so that the enzymes could be used repeatedly.
2. Description of the Prior Art
In the past, enzymes have been fixed or immobilized on both organic and inorganic supports. Enzymes can be immobilized on particles of various metal oxides so that the enzyme is bound or deposited on the surface of the oxide and is supported thereat to catalyze a specific reaction as shown for example in U.S. Pat. No. 3,850,751. This type of immobilization, while satisfactory, has the disadvantage that the enzyme will also desorb or release itself from the surface of the support over a period of time. The activity of the composite is thereby lowered over the long term, and the sample is contaminated with active enzyme which continues to react with the substrate after the sample has been removed from the reaction chamber of analysis. Accordingly, data for a given sample or batch may be obtained which may not be truly representative of the nature of the sample.
Many techniques have been attempted to lessen the rate at which enzymes desorb from a support structure. In U.S. Pat. No. 3,873,426, the enzyme and an organic dye, typically a halotriazine dye, are adsorbed onto alumina below pH 7. This code-position proposes to increase the affinity of the enzyme for the support. In such techniques, the enzyme and the added species, in this example the dye, can desorb into the test sample at some finite rate. Accordingly, another possible contaminant can be introduced in the test sample which gives false results when the sample is examined spectrophotometrically or electro-chemically.
U.S. Pat. No. 3,982,997 discloses the use of mixed metal oxides to immobilize enzymes and increase deposition of the enzyme and stability of the composite. While the technique does exhibit an increased initial activity of the enzyme, it does not bind the enzyme to the support which approximates an ideal strength of binding.
Many techniques have been utilized to covalently link an enzyme to the support to circumvent the desorption problems encountered in techniques as discussed above. These techniques of covalent bonding have generally used a coupling agent which bonds to the support by one reactive group and bonds to the enzyme by another reactive group. These groups have generally been reactive species such as diazos, silanes, and the like. For examples of these coupling agents reference is made to U.S. Pat. No. 3,930,951, and U.S. Pat. No. 3,519,538.
Many of these immobilization techniques have been used in large scale operation but have not been completely satisfactory. This is due to the expense in production of the composite, the lack of long-term stability of the composite, certain difficulties involved in purifying the coupling agents from the composite after its production, and in the gradual desorption of these additives into delicate samples or electrode systems used to monitor the product levels in the reactor.